klumpfuss distinguishes stem cells from progenitor cells during asymmetric neuroblast division

Abstract

Asymmetric stem cell division balances maintenance of the stem cell pool and generation of diverse cell types by simultaneously allowing one daughter progeny to maintain a stem cell fate and its sibling to acquire a progenitor cell identity. A progenitor cell possesses restricted developmental potential, and defects in the regulation of progenitor cell potential can directly impinge on the maintenance of homeostasis and contribute to tumor initiation. Despite their importance, the molecular mechanisms underlying the precise regulation of restricted developmental potential in progenitor cells remain largely unknown. We used the type II neural stem cell (neuroblast) lineage in Drosophila larval brain as a genetic model system to investigate how an intermediate neural progenitor (INP) cell acquires restricted developmental potential. We identify the transcription factor Klumpfuss (Klu) as distinguishing a type II neuroblast from an INP in larval brains. klu functions to maintain the identity of type II neuroblasts, and klu mutant larval brains show progressive loss of type II neuroblasts due to premature differentiation. Consistently, Klu protein is detected in type II neuroblasts but is undetectable in immature INPs. Misexpression of klu triggers immature INPs to revert to type II neuroblasts. In larval brains lacking brain tumor function or exhibiting constitutively activated Notch signaling, removal of klu function prevents the reversion of immature INPs. These results led us to propose that multiple mechanisms converge to exert precise control of klu and distinguish a progenitor cell from its sibling stem cell during asymmetric neuroblast division.

Fig. 1.

Neuroblasts prematurely differentiate in klu mutant brains. (A) Summary of the cell fate marker expression pattern in type I and II neuroblast lineages in Drosophila larval brains. GMC, ganglion mother cell; INP, intermediate neural progenitor; imm INP, immature INP; neurob, neuroblast; Pros, Prospero. (B-F) klu mutant brains show progressive loss of neuroblasts. (B-E) Brains were dissected from wild-type or klu^R51/09036 mutant larvae at 96 hours ALH and stained for the markers indicated. The white dotted line separates the central brain (left) from the optic lobe (right). Discs large (Dlg) marks the cell cortex. (F) Average type I and II neuroblasts per brain lobe in larvae of genotypes and stages indicated. Error bars indicate s.e.m. (G-L) Neuroblasts show reduced cell diameter and are likely to prematurely differentiate in klu mutant brains. Larvae carrying GFP-marked klu^+/+ or klu^−/− mosaic neuroblast clones (outlined by the yellow dotted line) were aged for 110 hours after clone induction and larval brains were stained for the markers indicated. (G-H‴) Type I neuroblast clones. (I-K‴) Type II neuroblast clones. (L) The frequency of klu^+/+ or klu^−/− clones containing neuroblasts of the cell diameter indicated. The following are indicated: type I neuroblast (Dpn⁺ Ase⁺), green arrow; GMC (Dpn⁻ Ase⁺), green arrowhead; type II neuroblast (Dpn⁺ Ase⁻), white arrow; Ase⁻ immature INP (Dpn⁻ Ase⁻), white arrowhead; Ase⁺ immature INP (Dpn⁻ Ase⁺), yellow arrow; INP (Dpn⁺ Ase⁺), yellow arrowhead. Scale bars: 20 μm in B-E; 10 μm in G-K‴.

Fig. 2.

Overexpression of klu induces supernumerary type II neuroblasts. (A-C) Klu is detected in neuroblasts but is undetectable in their immediate progenitor progeny. (A-B‴) Drosophila larvae carrying GFP-marked wild-type type I or II neuroblast lineage clones (outlined by the yellow dotted line) were aged for 72 hours after clone induction and larval brains were stained for the markers indicated. (C) Summary of the Klu expression pattern in type I and II neuroblast lineages. (D-E″) Overexpression of klu induces excess type II neuroblasts. Larvae of the genotype indicated were raised at 31°C for 72 hours ALH and larval brains were stained for the markers indicated. The white dotted line separates the central brain (left) from the optic lobe (right). (F-G‴) Overexpression of klu specifically induces supernumerary neuroblasts in type II neuroblast lineage clones. Larvae carrying GFP-marked type I or II lineage clones (outlined by the yellow dotted line) overexpressing klu were aged for 24 hours after clone induction and brains were stained for the markers indicated. Abbreviations and arrows/arrowheads as Fig. 1. Scale bars: 10 μm in A-B‴,F-G‴; 20 μm in D-E‴.

Fig. 3.

Misexpression of klu triggers the reversion of immature INPs to type II neuroblasts. (A-A‴) Overexpression of klu is not sufficient to trigger de-differentiation of INPs. Drosophila larvae carrying GFP-marked INP lineage clones (outlined by the yellow dotted line) overexpressing klu were aged for 24 hours after clone induction and brains were stained for the markers indicated. (B,C) Telophase neuroblasts overexpressing klu show asymmetric localization of apical and basal proteins. Phh3, phosphorylated histone H3. (D-F) The activity of erm-GAL4 is first detected in immature INPs. (D-E‴) Larvae expressing GFP driven by erm-GAL4 (II) or erm-GAL4 (III) were aged for 72 hours and brains were stained for the markers indicated. PointedP1 (PntP1) marks type II neuroblasts and Ase⁻ immature INPs. (F) Summary of the erm-GAL4 expression pattern in the type II neuroblast lineage. (G-J) Overexpression of klu in immature INPs leads to supernumerary type II neuroblasts. (G-I) Larvae overexpressing klu driven by erm-GAL4 were raised at 31°C for 72 hours ALH and brains were stained for the markers indicated. The white dotted line separates the central brain (left) from the optic lobe (right). (J) Average type II neuroblasts per brain lobe in larvae of the genotype indicated. 1×, 2× indicate the copy number of UAS-klu and erm-GAL4 (III) transgenes. Error bars indicate s.e.m. Abbreviations and arrows/arrowheads as Fig. 1. Scale bars: 10 μm in A-A‴,D-E‴; 5 μm in B,C; 20 μm in G-I.

Fig. 4.

Induction of supernumerary type II neuroblasts by Klu is dependent on the zinc-finger motifs. (A) The klu transgenes used in this study. (B-D) Drosophila larvae overexpressing various klu transgenes were raised at 31°C for 72 hours ALH and brains were stained for the markers indicated. The white dotted line separates the central brain (left) from the optic lobe (right). Phalloidin (Phall) marks the cell cortex. Type II neuroblasts (Dpn⁺ Ase⁻) are indicated (arrows). Scale bar: 20 μm. (E) Average type II neuroblasts per brain lobe in larvae of the genotype indicated. Error bars indicate s.e.m.

Fig. 5.

Brat suppresses reversion of immature INPs by antagonizing Klu. (A-C) Removal of klu function suppresses supernumerary type II neuroblasts and restores the formation of INPs and GMCs in brat strong hypomorphic mutant brains. (A-B⁗) brat^11/k06028 mutant Drosophila larvae carrying GFP-marked control (klu^+/+) and klu mutant type II neuroblast mosaic clones (outlined by the yellow dotted line) were aged for 72 hours after clone induction and brains were stained for the markers indicated. (C) Quantification of various cell types in the control and klu mutant clones in brat^11/k06028 mutant brains. (D-F) Co-expression of Brat suppresses Klu-induced supernumerary type II neuroblasts. (D-E⁗) Larvae carrying GFP-marked type II neuroblast lineage clones (outlined by the yellow dotted line) overexpressing klu or klu and brat were aged for 72 hours after clone induction and brains were stained for the markers indicated. (F) Average type II neuroblasts per brain lobe in larvae of the genotype indicated. Error bars indicate s.e.m. Arrows/arrowheads as Fig. 1. Scale bars: 10 μm.

Fig. 6.

Aberrant activation of Notch signaling induces reversion of immature INPs through klu. (A-D) Removal of klu function suppresses supernumerary type II neuroblasts induced by constitutively activated Notch signaling. (A-C⁗) Drosophila larvae carrying GFP-marked wild-type (klu^+/+) or klu^−/− type II neuroblast mosaic clones (outlined by the yellow dotted line) overexpressing Notch_intra were aged for 72 hours after clone induction and brains were stained for the markers indicated. (D) The frequency of clones containing one or more type II neuroblasts in larvae of the genotype indicated. (E-G) Overexpression of klu prevents Notch mutant type II neuroblasts from premature differentiation. (E-F⁗) Larvae carrying GFP-marked Notch mutant type II neuroblast mosaic clones (outlined by the yellow dotted line) alone or overexpressing klu were aged for 72 hours after clone induction and brains were stained for the markers indicated. (G) The frequency of clones containing one or no type II neuroblasts in larvae of the genotype indicated. (H) Model: Brat or Numb prevent the reversion of immature INPs to type II neuroblasts by antagonizing Klu. Abbreviations and arrows/arrowheads as Fig. 1. Scale bars: 10 μm.

Fig. 7.

Aberrant activation of Notch signaling induces reversion of GMCs in part through klu. (A-D) Co-expression of klu further exacerbates the formation of supernumerary type I neuroblasts induced by constitutively activated Notch signaling. (A-C⁗) Drosophila larvae carrying GFP-marked type I neuroblast lineage clones (outlined by the yellow dotted line) overexpressing klu, Notch_intra or klu and Notch_intra were aged for 48 hours after clone induction and brains were stained for the markers indicated. (D) Average type I neuroblasts per clone and the frequency of clones containing one or more type I neuroblasts in larvae of the genotype indicated. (E-G) Removal of klu function suppresses supernumerary type I neuroblasts induced by constitutively activated Notch signaling. (E-F⁗) Larvae carrying GFP-marked klu^+/+ or klu^−/− type I neuroblast mosaic clones (outlined by the yellow dotted line) overexpressing Notch_intra were aged for 72 hours after clone induction and brains were stained for the markers indicated. (G) Average type I neuroblasts per clone and the frequency of clones containing one or more type I neuroblasts in larvae of the genotype indicated. (H) Model: Numb prevents the reversion of GMCs to type I neuroblasts by antagonizing Klu. Abbreviations and arrows/arrowheads as Fig. 1. Scale bars: 10 μm.